Cell line for the expression of an alpha2delta2 calcium channel subunit

ABSTRACT

Described is a method for determining the binding ability of a compound to bond to an α2δ2 subunit of a calcium channel comprising: providing an α2δ2 subunit of a calcium channel, contacting the subunit with the compound, and determining the binding ability of the compound to bind to the subunit.

FIELD OF THE INVENTION

[0001] This invention pertains to cell lines which express an α2δ2subunit of a voltage-dependent calcium channel, where the cell lines mayalso express additional calcium channel subunits, and where the bindingof gabapentin, gabapentin analogues, pregabalin, or pregabalin to thecells may be determined.

BACKGROUND OF THE INVENTION

[0002] Voltage-dependent calcium channels have been linked tophysiological processes such as neurotransmitter release, secretion ofhormones, muscle contraction, and regulation of gene transcription. Afunctional channel requires at least three subunits, including the α1,α2δ, and β subunits. The channel may also contain a γ subunit. There areseveral known types of voltage-dependent calcium channels that have beendefined based on their electrophysiological characteristics andpharmacological properties. These types are L-, N-, P/Q-, R-, andT-type. Each type is primarily defined by its channel composition. Thetype of α1 subunit contained in the channel determines whether thechannel is an L-, N-, P/Q-, R-, or T-type channel. The activity of theα1 subunit is modulated by the α2δ and β subunits. Channel activity maybe further modulated by a fourth subunit, γ.

[0003] Molecular biological techniques have allowed elucidation of themechanism of voltage-dependent calcium channel action. Genes for each ofthe subunits have been isolated and cloned. There are currently nineknown genes encoding for different α1 subunits. The α1 subunit forms thepore which calcium ions flow through. The α1 subunit contains thevoltage sensor and is also responsible for the binding specificity ofcertain drugs or toxins that may be associated with the channel type.Channel current through the α1 pore may be modulated by association ofthe β, γ, or α2δ subunit. There are four known genes for theintracellular β subunit that may be differentially spliced. There aretwo known genes for the transmembrane γ subunit, one in skeletal muscleand a novel gene expressed in the brain. Only one isoform of α2δ wasinitially identified. Recently, however, two new α2δ genes wereidentified, α2δ2 and α2δ3. These genes have 55.6 and 30.3% homology withthe original α2δ1 gene (Klugbauer, et al., J. Neuroscience1999;19(2):684-691). The α2 and δ proteins are expressed by the samegene. The protein product is post-translationally cleaved, and the finalα2 and δ proteins are linked by disulfide bonds. The transmembrane δprotein secures the α2 protein to the cell membrane.

[0004] Studies have shown that the α2δ1 subunit contains a binding sitefor the anticonvulsant drug, gabapentin [1-(aminomethyl)cyclohexaneacetic acid] (Gee, et al., J. Biol. Chem. 1996;271(10):5768-5776).Gabapentin is a γ-aminobutyric acid (GABA) analogue. Gabapentin iseffective in the treatment of epilepsy and in decreasing seizurefrequency in both animal models and in human patients. The precisemechanism of action of gabapentin remains unclear. Recent experimentshave shown that gabapentin also binds to the α2δ2 subunit.

[0005] Functional channels may be formed by expression of the calciumchannel subunits in a cell. This technique is advantageous indetermining the effects of various molecules on channel action. U.S.Pat. No. 5,712,158 and U.S. Pat. No. 5,770,447 describe a stable cellline that is useful for investigating gabapentin binding properties tocalcium channel subunits. This cell line expresses the β subunit and theoriginal α2δ subunit (now referred to as α2δ1) at high levels.Transfecting the cells with any α1 subunit results in the formation offunctional calcium channels which can be used to evaluate the binding ofgabapentin and gabapentin-related compounds.

[0006] It is the object of this invention to provide a new cell linethat stably expresses a calcium channel α2δ2 subunit. It is a furtherobject of this invention to describe α2δ2 subtype-specific binding ofgabapentin, analogues of gabapentin, pregabalin, analogues ofpregabalin, and 3-alkyl derivatives of GABA.

SUMMARY OF THE INVENTION

[0007] The invention provides a method for determining the bindingability of a compound to an α2δ2 subunit of a calcium channelcomprising: providing an α2δ2 subunit of a calcium channel, contactingthe α2δ2 subunit with the compound, and determining the binding abilityof the compound to the α2δ2 subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 diagrams the molecular cloning of human α2δ2 into thepCDNA3.1 expression vector.

[0009]FIG. 2. RT-PCR Analysis of Human α2δ Tissue Distribution. One ngof double-stranded cDNA from different human tissues (CLONTECH) wasamplified by PCR with 35 cycles of 94° C. for 1 minute, 55° C. for 1minute, and 72° C. for 2 minutes. The generated PCR products representDNA fragments from nucleotide 958 to 2165 (hα2δ), 2534 to 3643 (hα2δ2),and 1920 to 3272 (hα2δ3).

[0010]FIG. 3. Northern Blot Analysis Human α2δ Tissue Distribution.Northern blotting was carried out as described in Materials and Methods.Human multiple tissue blots (CLONTECH) were hybridized withDigoxigenin-labeled cDNA synthesized from nucleotide 958 to 2165(hα2δ1), 2534 to 3643 (hα2δ2), and 1920 to 3272 (hα2δ3). The positionsof marker RNA are indicated to the left.

[0011]FIG. 4. Western Blot Analysis of Human and Mouse α2δ TissueDistribution. Membrane proteins from different human tissues (A, 0.5 μg)and mouse tissues (B, 100 μg) were loaded on 4% to 20% SDS-PAGE (NOVEX)and subjected to Western blot analysis (see Materials and Methods). Theblots were probed with anti-α2δ monoclonal antibody or polyclonalantibodies against δ2δ2 and α2δ3.

[0012]FIG. 5. Binding of [³H]Gabapentin to Membranes From COS-7 CellsTransfected With α2δ cDNA. COS-7 cells were transfected with 20 μg ofpcDNA3.1 (control), pcDNA3.1/porcine α2δ1 construct (pα2δ1), andpcDNA3.1//human α2δ2 construct (hα2δ2). The membranes were prepared for[³H]gabapentin binding assays (see Materials and Methods). Data are anaverage of three independent assays with triplet in each determination.The sane membranes (100 μg) were subjected to Western blot analysis withcorresponding antibodies as described in Materials and Methods.

[0013]FIG. 6. Disruption of Disulphide-Linkage Between α2 and δSubunits. An equal amount of membrane protein from each sample (0.5 μgfor pα2δ1 and 5 μg for hα2δ2) was incubated in the presence or absenceof 100 mM DTT for 10 minutes and resolved on a nonreducing SDS-PAGE andtransferred to a PVDF membrane. The blots were probed with either ananti-α2δ1 antibody (left) or an anti-α2δ2 antibody (right). Thepositions of marker proteins are indicated to the right.

[0014]FIG. 7. Scatchard Analysis of [³H]gabapentin (GBP) Binding toMembranes Form HEK293 Cells Overproducing Porcine α2δ1 (A) and Humanα2δ2 (B). The cell membranes were prepared from GKS02, a stable cellline for porcine α2δ1, and GKS07, a stable cell line for human α2δ2. Thespecific [³H]gabapentin binding was carried out as described inMaterials and Methods. The binding activity was expressed as pmole ofgabapentin bound per mg of protein. Each binding reaction contained 20μg of GKS02 membrane proteins or 10 μg of GKS07 membrane proteins. Datawere averages of three assays.

[0015]FIG. 8. Screening Cell Lines by [³H]Gabapentin (GBP) BindingActivity. HEK293 cells were transfected with human α2δ2. Single cloneswere selected by G418-resistance. “2923,” parental cells HEK293; “2L,”HEK293 cells stably expressing porcine α2δ1.

DETAILED DESCRIPTION OF THE INVENTION

[0016] As used herein, analogues of gabapentin include but are notlimited to alkyl-substituted gabapentin analogues, bridged gabapentinanalogues, and heterocyclic gabapentin analogues such as those describedby Bryans, et al. in J. Med. Chem. 1998;41:1838-1845. Analogues aredefined as “compounds with similar electronic structures but differentatoms” (Grant, et al., Chemical Dictionary, 5th ed., McGraw-Hill, 1987).Gabapentin has the structure:

[0017] Examples of gabapentin analogues are described in Bryans, et al.,supra, and include, but are not limited to:

[0018] A molecule with the structure:

[0019] This analogue is alkylated at position 3 on the cyclohexane ring.An analogue may be alkylated at any position on a carbon ring with analkyl group of from 1 to 4 carbon atoms. An analogue may also be amolecule with the structure:

[0020] This analogue is alkyl-substituted at the 3-position of thegabapentin ring. Molecules of this type include pregabalin

[0021] its analogues, and 3-alkyl derivatives of GABA.

MATERIALS AND METHODS

[0022] Porcine α2δ1 (pα2δ1) cDNA was from J. Brown (Brown J. P.,Dissanayke V. U. K., Briggs A. R., Milic M. R, Gee N., Anal. Biochem.,1998;255:236-243). Mouse α2δ3 (mα2δ3) cDNA was a generous gift from F.Hoffman (Klugbauer N., Lacinova L., Marais E., Hobom M., Hofmann F., J.Neurosci., 1999;19:684-691). Monoclonal antibody against α2δ1 waspurchased from Affinity Bioreagents, Inc. Polyclonal antibodies againstα2δ2 and α2δ3 were from Sandra Duffy (Pfizer). Human and mouse multipletissue blots and cDNA were purchased from CLONTECH. Mouse tissues werepurchased from Pel-Freez Biologicals. PCR reagents were from Invitrogen.ECL Western blot kit was from Armersham. Lipofectamine, growth media,restriction enzymes were from Life Technologies. HEK293 and COS-7 celllines were from ATCC. All other chemicals were from Sigma.

[0023] Cloning of Human α2δ2 Subunit. Human α2δ2 (hα2δ2) cDNA wasamplified by PCR from a human brain cDNA library. Based on the depositedDNA sequence of hα2δ2 subunit from GenBank (accession number AF042792),four overlapped DNA fragments, which covered the whole open readingframe of hα2δ2 cDNA from nt-14 to 994 (fragment H), 845 to 1816(fragment F), 1517 to 2791 (fragment D), and 2681 to 3790 (fragment C),were generated by PCR and then cloned into expression vector pcDNA3.1 byTA cloning kit. The sequences of the primer pairs used were:

[0024] 5′-TCTTGAATGGAAACATGGCGGTGC-3′ (SEQ ID No. 1) and

[0025] 5′-TATACCAGGGTCTCCTTCGGACAT-3′ (SEQ ID No. 2) (fragment H);

[0026] 5′-ATGTGTTCATGGAAAACCGCAGAC-3′ (SEQ ID No. 3) and

[0027] 5′-AGCCGTTCAGGTCAATGGCAAACA-3′ (SEQ ID No. 4) (fragment F);

[0028] 5′-CCATCCGCATCAACACACAGGAAT-3′ (SEQ ID No. 5) and

[0029] 5′-GTAAGTCCTCATTGTTAACCTCGC-3′ (SEQ ID No. 6) (fragment D);

[0030] 5′-CTGAGAAGTTCAAGGTGCTAGCCA-3′ (SEQ ID No. 7) and

[0031] 5′-GATGTGATTTGGGTGCCAAACACC-3′ (SEQ ID No. 8) (fragment C). Thefour fragments were cut at internal unique restriction enzyme sites atnt 791 (Pf1M I), 1395 (Xba I), and 2628 (Hpa I), and assembled intopcDNA3.1 vector (Invitrogen, Carlsbod, Calif.) at Hind III/Xho I sites(see FIG. 1).

[0032] RT-PCR. Double-stranded cDNA preparations from different tissues(CLONTECH) were used for PCR reaction with 35 cycles at 94° C. for 1minute, 55° C. for 1 minute, and 72° C. for 2 minutes. The reactionswere performed in a solution containing 1 ng cDNA, 10 pM primers, 1 mMdNTPs, and 1×PCR buffer in a volume of 50 μL. Ten microliters of thereaction mix was loaded on 1% agarose gel. The primer pairs for humanα2δ1, α2δ2, and α2δ3 were

[0033] 5′-GACGCGGTGAATAATATCACAGCC-3′ (SEQ ID No. 9) and

[0034] 5′-ACAAATCGTGCTTTCACTCCCTTG-3′ (nt 958 to 2165; accession numberM76559) (SEQ ID No. 10);

[0035] 5′-CTGAGAAGTTCAAGGTGCTAGCCA-3′ (SEQ ID No. 11) and

[0036] 5′-GATGTGATTTGGGTGCCAAACACC-3′ (nt 2534 to 3643; accession numberAF042792) (SEQ ID No. 12); and

[0037] 5′-CGTGTCCTTGGCAGATGAATGGTC-3′ (SEQ ID No. 13) and

[0038] 5′-CATCTCAGTCAGTGTCACCTTGAG-3′ (nt 1920 to 3272; accession numberAJ272213) (SEQ ID No. 14), respectively. The expected lengths of PCRproducts from human α2δ1, α2δ2, and α2δ3 were 1208, 1110, and 1352 bp.These primers were specific for each subtype of α2δ as determined bysequencing analysis of the corresponding PCR products.

[0039] Northern Blot Analysis. Multiple Tissue Northern Blots (CLONTECH)were hybridized and washed according to the manufacturer'srecommendation. Digoxigenin-labeled probes specific for subtypes of α2δwere generated by PCR and hybridized in 10 mL EasyHyb (BoehringerMennhaim) at 50° C. overnight. The same pairs of primers as those usedfor RT-PCR were employed to generate the probes. The blots were washedtwice, first in 2×SSC and 0.1% SDS at room temperature for 5 minutes,then in 0.1×SSC and 0.1% SDS at 68° C. for 15 mninutes. Detection ofexpression was in accordance with the manufacturer's instructions(Boehringer Mennhaim).

[0040] Cell Culture and Transfection. COS-7 and HEK293 cells werecultured in DMEM and RPMI 1640 media, respectively. The media weresupplemented with 50 units/mL penicillin, 50 μg/mL streptomycin, and 10%heat-inactivated fetal bovine serum (FBS), in a humidified incubatorwith 95% air and 5% CO₂ at 37° C. For transient transfection into COS-7cells, 20 μg of plasmid DNA (vector or the same vector with α2δ insert)was incubated with 30 μL of lipofectamine. The mixture was overlaid ontothe cells in 1.5 mL serum-free medium and incubated for 5 hours. ThenFBS was added to the dishes to bring the final concentration to 10%. Themedium was changed next morning. Forty-eight hours after thetransfection, the cells were harvested for membrane preparation. Forstable transfection of porcine α2δ1 and human α2δ2 into HEK 293 cells,the same procedure was applied as that for a transient transfectionexcept for that 800 μg/mL G418 (gentacin) was added to the cells 48hours after the transfection. Two clones, GKS02 and GKS07, showedhighest expression of porcine α2δ1 and human α2δ2, respectively, andwere selected for further binding studies. The cell line has ATCC No.PTA-1823. In addition, hosts for expression of α2δ2 protein bindingassays can also include eukaryotic expression systems such as yeast,insect cells, and mammalian cells (CHO, COS-7, HEK293, etc.).

[0041] Membrane Preparation. Membranes were prepared from tissues orcultured cells. The cells were washed twice with cold PBS and thenscraped off with cold buffer containing Tris (5 mM, pH 7.4), EDTA (5mM), PMSF (0.1 mM), leupeptin (0.02 mM), and pepstatin (0.02 mM). Thecells were incubated on ice for 30 minutes, followed by sonication for30 to 40 seconds. For membrane preparations from tissues, the tissueswere sliced into small pieces and subjected to sonication at interval of10 seconds 4 times. The resulting homogenates from tissues or culturedcells were centrifuged for 10 minutes at 750 to 1000×g, and then thesupernatants were centrifuged at 50,000×g for 30 minutes. The resultingpellets were resuspended in the same buffer as described above.

[0042] Western Blot Analysis. The cell membranes (0.5 μg for GKS07cells, 5 μg for GKS02 cells, 100 μg for transiently transfected cells ortissues) were resolved by 4% to 20% SDS-PAGE and transferred tonitrocellulose membranes using semi-dry transferring unit. The membraneswere incubated with either rabbit anti-α2δ1, α2δ2, and α2δ3 antibodiesfor 1 hour at room temperature, followed by washing with 1×PBS. Theblots were incubated with anti-rabbit IgG for 1 hour and developed withECL reaction according to the procedure recommended by manufacturer.

[0043] Binding Assays. The radioligand-binding assay was done usingmembrane proteins incubated in the presence of 20 nM [³H]gabapentin. Themembranes (100 μg of proteins for transiently transfected cells, 20 μgfor GKS02 cell membranes, and 10 μg for GKS07 cell membranes) wereincubated in 10 mM HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (pH 7.4) for40 to 50 minutes at room temperature, and then filtered onto pre-wettedGF/C membranes and quickly washed five times with 3 mL of ice cold 50 mMTris buffer (pH 7.4). The filters were dried and counted in a liquidscintillation counter. For determining nonspecific binding, the bindingassays were performed in the presence of 10 μM pregabalin (Gee N S.,Brown J. P., Dissanayake V. U., Offord J., Thurlow R., Woodruff G. N.,J. Biol. Chem., 1996;271:5768-5776). The specific binding was obtainedby subtracting nonspecific binding from the total binding. Clone #7 wasidentified as the highest α2δ2 subunit expressing clone. Binding assayscan also be performed using recombinant and/or purified α2δ2 proteinfrom human and other mammalian species, for screening α2δ2subtype-selective inhibitors.

Results

[0044] Tissue Distribution of α2δ Transcripts. Tissue distribution ofhα2δ1, hα2δ2, and hα2δ3 mRNA was first examined by RT-PCR analysis.These probes were designed to specifically amplify three subtypes ofα2δ. As shown in FIG. 2, single PCR products corresponding well to thepredicted sizes of hα2δ1, hα2δ2, and hα2δ3 (1208, 1110, and 1352 bp)appeared in almost all tissues tested. A much higher level of hα2δ2transcript was found in lung than any other tissues including brain.Since the PCR products showed sequences identical to the correspondingα2δ, the wide scope of tissue distribution revealed the ubiquitousfeature of hα2δ mRNA expression. However, the RT-PCR condition used heredid not yield quantitative estimation of α2δ mRNA levels among differenttissues, Northern analysis is necessary for estimating the relativeabundance of each subtype of hα2δ mRNA. Northern blots demonstrated thatall three hα2δ genes were expressed about equally well in brain, heart,and skeletal muscle except for the much higher expression of hα2δ2 inskeletal muscle (FIG. 3). In addition to these three tissues, the mostabundant hα2δ1 transcript was found in lung. The highest expression ofhα2δ2 mRNA in lung was consistent with the above described RT-PCRresults and also agreed well with one recent report (Gao B., Sekido Y.,Maxinov A., Saad M., Forgacs E., Latif F., et al., J. Biol. Chem.,2000;275:12237-12242), but differed from an early observation (KlugbauerN., Lacinova L., Marais E., Hobom M., Hofmann, F., J. Neurosci.,1999;19:684-691). In the present study we also detected a small amountof hα2δ1 and hα2δ3 mRNAs in liver and kidney, respectively. Results fromthis and other laboratories (Klugbauer, Supra., 1999; Gao, Supra., 2000,and our unpublished data) have shown that expression of mouse α2δ3(mα2δ3) is restricted to the brain. The expression of hα2δ3 also intissues other than brain suggested species difference in α2δ3expression.

[0045] In the brain, hα2δ1, hα2δ2, and hα2δ3 were detected in everyportions of brain tissues tested including cerebellum, cerebral cortex,medulla, occipital pole, frontal lobe, temporal lobe, and putamen. Ahigher level of hα2δ2 transcript was found in cerebellum than cerebralcortex, while reverse was true for hα2δ3. For hα2δ1, its mRNA wasapproximately equally distributed in these two regions. The expressionpatterns of the three isoforms in these two brain regions were inaccordance with previous in situ hybridization results (Klugbauer,Supra., 1999; Hobom M., Dai S., Marais E., Lacinova L., Hofmann F.,Klugbauer N., Eur. J Neurosci., 2000;12:1217-1226). In addition, allthree subtypes of α2δ mRNA were found in spinal cord, but at lowerlevels than that found in the brain.

[0046] Tissue Distribution of α2δ proteins. Although the level ofprotein is function of the steady-state level of mRNA, the relativeabundance of mRNA and protein of specific gene is not alwaysproportional, which may reflect post-transcriptional regulation (JacksonV. N., Price N. T., Carpenter L., Halestrap A. P., Biochem. J.,1997;324:447-453). To examine the relative levels of human and mouse α2δsubunits across tissues, we used antibodies raised against specificsubtypes of α2δ protein for Western analysis. Equal amounts of proteinswere loaded on SDS polyacrylamide gels. Consistent with the ubiquitousdistribution of hα2δ1, Western blots of human and mouse tissues showedthat both hα2δ1 and mα2δ1 proteins were widely distributed, althoughhα2δ1 in lung and jejunum were not detectable. By contrast, hα2δ3protein was only detected in brain, not in lung, testis, aorta, spleen,jejunum, and kidney (FIG. 4A). Similarly, mα2δ3 protein was found onlyin brain, not in heart, kidney, liver, lung, pancreas, stomach, spleenthymus, ovary, pituitary, thyroid, and prostate. Surprisingly, incontrast to predominant expression of hα2δ2 transcript in lung (FIGS. 2and 3), hα2δ2 protein was predominantly found in brain and the level ofhα2δ2 protein was not detectable in lung (FIG. 4A). In addition tobrain, low levels of hα2δ2 protein were also found in aorta, testis, andventricular muscle. There seemed to be two immunoreactive bands intestis with one equivalent to predicted molecular weight of hα2δ2 (175kDa) and the other showing slightly lower molecular weight. This lowermolecular protein appeared to be similar to the predominant banddetected in ventricular muscle. As previously observed with pα2δ1, thislower band may represent the dissociated α2 subunit from the α2δ proteinor an isoform of α2δ2 (Brown J. P., Dissanayke V. U. K., Briggs A. R.,Milic M. R, Gee N., Anal. Biochem., 1998;255:236-243; Wang M., OffordJ., Oxender D. L., Su, T. Z., Biochem. J., 1999;342:313-320). Inaddition, two immunoreactive bands were also detected in mouse heart byanti-α2δ2 antibodies, but the predominant band in this case hadmolecular weight higher than that found in other tissues (FIG. 4B).

[0047] Disulphide Linkage of α2 and δ Proteins. It has been shown thatα2 and δ subunits of α2δ1 were linked by disulphide bond (Wang, Supra.,1999). Since the amino acid sequence in δ region is less conservedbetween α2δ1 and α2δ2, it is interesting to know if α2δ2 protein is alsocleaved into two subunits post translation. To examine such apossibility, cell membranes from HEK 293 cell lines overproducing pα2δ1(GKS02) and hα2δ2 (GKS07) proteins were treated or untreated with 100 mMDTT before gel electrophoresis. In the presence of DTT, both pα2δ1 andhα2δ2 proteins were shifted to a position predicted for α2, suggestingthat as with pα2δ1, hα2δ2 also consists of two subunits that are linkedby disulphide bond (FIG. 6).

[0048] [³H]Gabapentin Binding. To determine the gabapentin bindingproperties of the cloned hα2δ2, membranes were isolated from COS-7 cellstransiently transfected with pα2δ1, hα2δ2, and vector pcDNA3.1.Expression of the corresponding α2δ proteins was examined by Westernblots. As shown in FIG. 5, transfection of the cells with pα2δ1 resultedin a prominent increase in gabapentin binding. Similarly, the cellsexpressing hα2δ2 exhibited about fourfold increase in gabapentin-bindingactivity. Although a slightly increased binding activity was observed inthe cells transfected with pcDNA3.1 vector alone, statistic analysis didnot show that this smaller change was significant.

[0049] Gabapentin binding K_(D) and the binding properties of pα2δ1 andhα2δ2 were determined in cell lines GSK02 (pα2δ1) and GKS07 (hα2δ2). InHEK293 cells stably expressing pα2δ1, [³H]gabapentin bound to a singlepopulation of sites as demonstrated in previous report (Gee, Supra.,1996) with K_(D) value of 72±9 nM (FIG. 7A). Similarly, a singlepopulation of binding sites were also observed in hα2δ2-containingmembranes (FIG. 7B), but the K_(D) value was higher than that of pα2δ1(156±25 nM). To determine pharmacological properties of hα2δ2, severalcompounds were selected for competition with [³H]gabapentin binding. Asimilar, but not identical profile of competition was seen in the twosubtypes of α2δ protein (Table 1). For instance, binding to bothsubtypes of α2δ were stereo-selective because L-leucine was markedlymore potent than its D-enantiomer. The affinities of BCH, a modelsubstrate of system L transport (Su T. Z., Lunney E., Campbell G.,Oxender D. L., J. Neurochem., 1995;64:2125-2131), and phenylalanine wereweak for both subtype proteins. On the other hand, gabapentin binding toα2δ2 was more sensitive to (S+)-3-isobutyl GABA (pregabalin) with IC₅₀value of 96 nM as compared to 149 nM for pα2δ1. TABLE 1 IC₅₀ Values forInhibition of [³H]Gabapentin Binding to Membranes From Stable Cell LinesOverproducing Porcine α2δ1 (GKS02) and Human α2δ2 (GKS07) by SelectedAmino Acids Compounds GKS02 (pα2δ1) GKS07 (hα2δ2) Gabapentin 132 282Pregabalin 149 96 L-leucine 118 205 L-phenylalanine 825 2,960 D-leucine198,960 151,510 BCH 1,028 775

[0050]FIG. 8 also illustrates the screening of stable cell lines thatexpress human α2δ2 protein.

[0051] While the forms of the invention herein disclosed constitutepresently preferred embodiments, many others are possible. It is notintended herein to mention all of the possible equivalent forms orramifications of the invention. It is understood that the terms usedherein are merely descriptive, rather than limiting, and that variouschanges may be made without departing from the spirit or scope of theinvention.

1 14 1 24 DNA Homo sapiens 1 tcttgaatgg aaacatggcg gtgc 24 2 24 DNA Homosapiens 2 tataccaggg tctccttcgg acat 24 3 24 DNA Homo sapiens 3atgtgttcat ggaaaaccgc agac 24 4 24 DNA Homo sapiens 4 agccgttcaggtcaatggca aaca 24 5 24 DNA Homo sapiens 5 ccatccgcat caacacacag gaat 246 24 DNA Homo sapiens 6 gtaagtcctc attgttaacc tcgc 24 7 24 DNA Homosapiens 7 ctgagaagtt caaggtgcta gcca 24 8 24 DNA Homo sapiens 8gatgtgattt gggtgccaaa cacc 24 9 24 DNA Homo sapiens 9 gacgcggtgaataatatcac agcc 24 10 24 DNA Homo sapiens 10 acaaatcgtg ctttcactcc cttg24 11 24 DNA Homo sapiens 11 ctgagaagtt caaggtgcta gcca 24 12 24 DNAHomo sapiens 12 gatgtgattt gggtgccaaa cacc 24 13 24 DNA Homo sapiens 13cgtgtccttg gcagatgaat ggtc 24 14 24 DNA Homo sapiens 14 catctcagtcagtgtcacct tgag 24

What is claimed is:
 1. A method for determining the binding ability of acompound to a cell expressing an α2δ2 subunit of a calcium channelcomprising: providing a cell line expressing an α2δ2 subunit of acalcium channel; contacting the cells with the compound; and determiningthe binding ability of the compound to the cells.
 2. The method of claim1 wherein the compound is gabapentin.
 3. The method of claim 1 whereinthe compound is a gabapentin analogue.
 4. The method of claim 3 whereinthe gabapentin analogue is alkylated at any position on a carbon ringwith an alkyl group of from 1 to 4 carbon atoms.
 5. The method of claim3 wherein the gabapentin analogue is a 3-alkyl substitution ofgabapentin.
 6. The method of claim 1 wherein the compound is pregabalin.7. The method of claim 1 wherein the compound is a 3-alkyl derivative ofγ-aminobutyric acid (GABA).
 8. A stable cell line expressing an α2δ2subunit of a calcium channel.
 9. The cell line of claim 8 having ATCCNo. PTA-1
 823. 10. A method for determining the binding ability of acompound to an α2δ2 subunit of a calcium channel comprising: providingan α2δ2 subunit of a calcium channel; contacting the α2δ2 subunit withthe compound; and determining the binding ability of the compound to theα2δ2 subunit.
 11. The method of claim 10 wherein the compound isgabapentin.
 12. The method of claim 10 wherein the compound isgabapentin analogue.
 13. The method of claim 12 wherein the gabapentinanalogue is alkylated at any position on a carbon ring with an alkylgroup of from 1 to 4 carbon atoms.
 14. The method of claim 12 whereinthe gabapentin analogue is a 3-alkyl substitution of gabapentin.
 15. Themethod of claim 10 wherein the compound is pregabalin.
 16. The method ofclaim 10 wherein the compound is a 3-alkyl derivative of GABA.
 17. Themethod of claim 10 wherein the α2δ2 subunit is a purified protein. 18.The method of claim 10 wherein the α2δ2 subunit is a recombinantprotein.